Composition comprising the extract of herbs for preventing or treating neurodegenerative disorders

ABSTRACT

This invention relates to a pharmaceutical composition and a health functional food for preventing or improving neurodegenerative disorders comprising mixed herb extracts of  Dioscorea Rhizoma  and  Dioscorea nipponica  in a weight ratio of 3.5:1 (w/w). The herb extracts mixed  Dioscorea Rhizoma  and  Dioscorea nipponica  in a weight ratio of 3.5:1 have the synergetic effects on increasing the amount of nerve growth factor in vivo, increasing the neural cell proliferation, promoting the formation of neuritis and enhancing cognitive abilities. Thus, the herb extracts of the present invention may be used for a pharmaceutical composition and a health functional food for preventing or treating neurodegenerative disorders.

TECHNICAL FIELD

This invention relates to a composition comprising the extract of herbsfor preventing or treating neurodegenerative disorders, wherein itcomprises Dioscorea Rhizoma and Dioscorea nipponica mixed in a weightratio of 3.5:1 (w/w).

ACKNOWLEDGEMENT

This work was supported by the Global Leading Technology Program of theOffice of Strategic R&D Planning (OSP) funded by the Ministry ofKnowledge Economy, Republic of Korea. (No. 10039303)

BACKGROUND ART

Neurodegenerative disorders mean a gradually structural and functionalloss of a nerve cell (neuron). They usually affect a particular part ofthe nervous system, accompanying the symptoms such as dementia,extrapyramidal disease, cerebellar disorder, dysesthesia or dyskinesia.The complex symptoms may be shown when affecting the various parts atthe same time. A diagnosis is gotten by a patient's clinical sign.However, the diagnosis in this case is difficult to make because itshows multiple symptoms and the varied diseases have the common clinicalsigns. (Soc. Sci. Med. Vol. 40. No. 6, pp. 847-585, 1995)

Signs of the onset of neurodegenerative disorders appear gradually andmostly occur with aging. Once an outbreak, neurodegenerative disordersprogress for several years or decades until death. It is known that thegenetic effects according to a family history are considerable.According to the clinical symptoms, degenerative disorders areclassified into paralytic dementia (Alzheimer's disease etc.),neurologic disorder (Pick's disease etc.), abnormalities in posture andexercise (Parkinson's disease etc.), progressive ataxia, muscularatrophy and weakness, and sensation and movement disorders.(International Journal of Engineering and Technology, Vol.2, No.4,August 2010 Classification of Neurodegenerative Disorders Based on MajorRisk Factors Employing Machine Learning Techniques)

In 1980s, it was raised that neurotrophic factors have the potential totreat neurodegenerative disorders as Alzheimer's disease experiment(Nature. 1987 Sep 3-9; 329(6134):65-8. Amelioration of cholinergicneuron atrophy and spatial memory impairment in aged rats by nervegrowth factor) The loss of neuron of basal forebrain by aging known asAlzheimer's disease is recovered by administration of nerve growthfactor (NGF) to the lateral ventricle of the brain. As the memoryimprovement of tested animals is reported, the studies for treatingneurodegenerative disorders are conducted by using neurotrophic factor.It met with a good result that the function of motor neurons damaged byBrain-derived neurotrophic factor (BDNF), Neurotrophin-3 (NT-3),Neurotrophin-4 (NT-4), and Ciliary neurotrophic factor (CNTF) asneurotrophic factor family is recovered in the study after hurting thefunction of the motor neruons by sectioning the facial nerves and thesciatic nerves as the follow-up study. (Nature. 1992 Dec. 24-31;360(6406):757-9. Brain-derived neurotrophic factor prevents the death ofmotor neurons in newborn rats after nerve section.). In the experimentused gene recombination mice (wobbler) which suffered from losing itsmotor neurons and function gradually, the function was enhanced byadministrating BDNF and CNTF to increase the number of motor neurons.(Science. 1994 Aug. 19; 265(5175):1107-10. Arrest of motor neurondisease in wobbler mice cotreated with CNTF and BDNF). Besides the saidexperiments, neurotrophic factors increase neurons and their function inthe pathological model of motor neurons and various senses, so it showedthe improvement of disorder related to memory, perception, and behaviorin the laboratory animals.

Based on the results of pre-clinical experiments, there were trials toapply neurotrophic factor for the treatment of Lou Gehrig's disease in1990s. Lou Gehrig's disease is a degenerative nervous disease which onlymotor neurons die out selectively and makes human die due to thedysfunction of respiratory organs with paralysis of the whole body. BDNFwas hypodermically or subarachnoidally administered, however, a pain ofinjection region and the side effect of digestive system was shown. So,there had been no choice but to administer the smaller amount of BDNFthan the pre-clinical experiment. As a result, the regeneration andimprovement of motor neuron and its function had minimal effect. (ExpNeurol. 1993 November;124(1):64-72. Review. Experimental rationale forthe therapeutic use of neurotrophins in amyotrophic lateral sclerosis)Similarly, symptoms such as a fever, a pain of injection region or aloss of appetite which were more severe adverse reactions than the caseof administration of BDNF were shown when CNTF was injected to thepatient suffered from Lou Gehrig's disease, therefore CNTF wasadministered with a limited amount. As a result, the regeneration andimprovement of motor neuron and its function was insignificant.(Neurobiol Aging. 1994 March-April;15(2):249-51. Review Neurotrophicgrowth factors and neurodegerenative diseases: therapeutic potential ofthe neurotrophins and ciliary neurotrophic factor) The method of havingneurotrophic factor such as BDNF, CNTF, etc as a form of the recombinantprotein reach the central and peripheral nervous system by injecting invivo had the limited amount of injecting protein. In case of theexperiment which attempted to treat the patient having Alzheimer'sdisease using NGF, it couldn't show the significant result since therewere side effects, the limit of injecting amount, the drug delivery anduncertain pharmacodynamics.

Although a disappointing result of the clinical experiment, there havebeen many experimental evidences which are possible to treatneurodegenerative disorders using neurotrophic factors. Typically, incase of Huntington's disease, which its symptoms are abnormal movements,personality changes, decreased cognitive ability, and early death due toincreasing poly Q in Huntington protein, a lot of studies noted BDNF asa main target of abnormal Huntington protein. This theory was supportedby decreasing the amount of BDNF in the striatum of the pathologicallaboratory animals and the patient having Huntington's disease.(Science. 2001 Jul. 20;293(5529):493-8. Epub 2001 Jun. 4 Loss ofHuntington-mediated BDNF gene transcription in Huntington's disease)

By the way, when the method to administer neurotrophic factorhypodermically or subarchinoidally in vivo with the form of arecombinant protein as same as the existing method in order to treatdegenerative disorders was chosen, decreasing the amount ofadministration and reducing its effect were repeated due to the sideeffect. Therefore the studies of neurotrophic factor enhancer which isindirect way to increase the amount of neurotrophic factorbiosynthesized itself in vivo in 2000s. (Hum Gene Ther. 1999 Dec.10;10(18):2987-97. Brain-derived neurotrophic factor-mediated protectionof striatal neurons in an excitotoxic rat model of Huntington's disease,as demonstrated by adenoviral gene transfer)

Dioscorea Rhizoma, a plant belonging to Dioscoreaceae, is the term forfresh rhizome of Dioscorea batatas Decaisne or Dioscorea japonicaThunberg, void of periderm,or for that obtained after the fresh rhizomewas steamed and dried in the herbal medicine. It is widely distributedin Korea, China, and Japan, and was used as a medicine. It has remedialeffects on tonic nutrition, digestive disorders, diabetes, cough,pulmonary disease, strengthening kidney function, etc and toxicity andside effects often have not released. Moreover, Dioscorea nipponica is arhizome of Dioscorea nipponica Makino, a climbing perennial plant ofDioscoreaceae. It is widely distributed in Korea, China, and Japan, andwas used as a medicine. It has effects on better circulation of blood,loosening the muscles, removing indigestion, keeping one s urine open,removing sputum, and prevention of malaria paroxysm and toxicity andside effects often have not released.

Korean Patent No. 854621 provides a composition for the prevention andtreatment of peripheral neuropathy, comprising an extract from a plantselected from among Dioscorea nipponica, Dioscorea quinqueoloba,Dioscorea batatas, Dioscorea japonica and Dioscorea tokora, disclosingthat the composition induces the growth of neurites and increases thesecretion of endogenous nerve growth factor, thus being effective forpreventing or treating peripheral neuropathy.

The present inventors confirmed that the function of the extract from aplant selected from among Dioscorea nipponica, Dioscorea quinquoloba,Dioscorea batatas, Dioscorea japonica and Dioscorea tokora was to inducethe significant growth of neurites and to increase the secretion ofendogenous NGF as disclosed in Korean Patent No. 854621. Base on that,the present invention has been completed after ascertaining thatDioscorea Rhizoma and Dioscorea nipponica in the selectively particularratio show very significant synergism although the said herbs each ortheir mixed herb extracts surprisingly induce almost same growth ofneurites as well as increase the secretion of endogenous nerve growthfactor while studying a herb extract which has effects on neural cellproliferation, promotion of neuritis formation and enhancing cognitiveabilities by increasing the contents of nerve growth factor in thelaboratory animals in vivo.

DISCLOSURE OF INVENTION Technical Problem

It is therefore an object of the present invention to provide anoptional herbal composition which shows the synergy to enhance cognitiveabilities by increasing the neural cell proliferation and promoting theformation of neuritis after stimulating the production and secretion ofnerve growth factors, among the herbs disclosed in Korean Patent No.854621, to enhance the regeneration and prevent apoptosis of neurons.

It is another object of the present invention to provide apharmaceutical composition and a health functional food for preventingor treating neurodegenerative disorders, comprising the said herbcomposition as an active ingredient.

Solution to Problem

In accordance with an aspect of the present invention, there is provideda pharmaceutical composition and a health functional food for preventingor treating neurodegenerative disorders comprising a mixed extract ofDioscorea Rhizoma and Dioscorea nipponica in a weight ratio of 3.5:1(w/w).

Hereinafter, the present invention will be described in detail.

The present invention relates to a pharmaceutical for preventing ortreating neurodegenerative disorders comprising a mixed extract ofDioscorea Rhizoma and Dioscorea nipponica in a weight ratio of 3.5:1(w/w).

The mixed extract of Dioscorea Rhizoma and Dioscorea nipponica in aweight ratio of 3.5:1 (w/w) compared to the total weight is a crudeextract, which is an extract preferably extracted with 50% of ethanol.

The extracts of Dioscorea Rhizoma and Dioscorea nipponica may beobtained as below. First, herbs were prepared by cutting dried herbsafter cleaning and drying Dioscorea Rhizoma and Dioscorea nipponicarespectively. Then, the mixed herb extracts of Dioscorea Rhizoma andDioscorea nipponica in a weight ratio of 3.5:1 (w/w) could be obtainedby concentration under reduced pressure after one time of coldextraction for 48 hours in a room temperature with 50% of ethanol 5times more than the total weight of the said cutting herbs.

The present invention provides the use of herb extracts mixed withDioscorea Rhizoma and Dioscorea nipponica in a weight ratio of 3.5:1(w/w) for preparing a pharmaceutical composition for preventing ortreating neurodegenerative disorders.

The examples of neurodegenerative disorders by the present inventioninclude Alzheimer's disease, Creutzfeldt-Jakob disease, Huntington'sdisease, multiple sclerosis, Guillain-Barre syndrome, Parkinson'sdisease, Lou Gehrig's disease, paralytic dementia caused by gradualnerve cell death and diseases caused by progressive incotinentia.

According to the present invention, a pharmaceutical preparation forpreventing or treating neurodegenerative disorders can be formulated byadding a pharmaceutically acceptable carrier, diluent or diluting agentto the mixed herb extracts of Dioscorea Rhizoma and Dioscorea nipponicain a weight ratio of 3.5:1 (w/w).

The herb extracts needed to formulate a pharmaceutical preparation ofthe present invention include 0.01 to 80% of the said extracts,preferably 1 to 50% by weight compared to the total weight.

The carrier, diluent or diluting agent which can be included in thecomposition of the present invention may be lactose, dextrose, sucrose,sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum Acacia,alginate, gelatin, calcium phosphate, calcium silicate, cellulose,methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone,water, methylhydroxybenzoate, prophylhydroxybenzoate, talc, magnesiumstearate or mineral oil.

Furthermore, the composition of the present invention can be formulatedand used in forms of oral agent such as powder, granules, tablets,capsules, suspension, emulsion, syrup or aerosol, external preparation,suppository or sterile injecting solution according to the ordinarymethod.

Specifically, a pharmaceutical preparation could be prepared by usingdiluents or diluting agents such as filter, extender, binding agent,moistening agent, disintegrating agent or surfactant which are commonlyused. The solid pharmaceutical preparation for oral administrationincludes tablets, pills, powder, granules and capsules. The solidpharmaceutical preparation can be formulated by adding at least one ormore diluting agents such as starch, calcium carbonate, sucrose, lactoseor gelatin to the said herb complex. Moreover, lubricants such asmagnesium stearate or talc aside from the said diluting agents. Theliquid pharmaceutical preparation includes suspension, liquid medicinefor internal use, emulsion or syrup. It could include the variousdiluting agents such as moistening agent, sweetening agent, airfreshener or preserved agent aside from the simple diluents such aswater and liquid paraffin which are commonly used. A pharmaceuticalpreparation for parenteral administration includes sterile solution,nonaqueous solvent, suspension, emulsion, lyophilized agent orsuppository. Prophyl glycol, polyethylene glycol, vegetable oil likeolive oil or injectable ester like ethylolate could be used fornonaqueous solvent or suspension. For suppository, witepsol,polyethylene glycol, tween 61, cacao butter, laurinum orglycerol-gelatin could be used.

In accordance with another aspect of the present invention, there isprovided a method for treatment of neurodegenerative disorders, whereina pharmaceutical composition comprising the herb extracts mixed withDioscorea Rhizoma and Dioscorea nipponica in a weight ratio of 3.5:1 asan active ingredient is administered to mammals including human with apharmaceutically active amount.

The dosage of the pharmaceutical composition comprising the herbextracts mixed with Dioscorea Rhizoma and Dioscorea nipponica in aweight ratio of 3.5:1 depends on age, sex and weight of a patient,however, can be administered with an amount of 0.01 to 10 g/kg,preferably 1 to 5 g/kg which amount is administered once a day ordivided into many times a day. Also, the dosage can be increased ordecreased according to injection route, degree of disease, sex, weight,age, health condition, diet, injection time, injection method orexcretion rate. Therefore the dosage is in no way intended to limit thescope of the present invention.

The pharmaceutical composition comprising the herb extracts mixed withDioscorea Rhizoma and Dioscorea nipponica in a weight ratio of 3.5:1(w/w) of the present invention may be administered to mammals such asrats, mice, cattle or human through the various routes. All the way ofadministration may be expected, for example, oral, rectal or vein,intramuscular, hypodermic, endometrial or intracerebroventicularinjections.

The herb extracts mixed with Dioscorea Rhizoma and Dioscorea nipponicain a weight ratio of 3.5:1 of the present invention has almost nevershow toxicity or side effects, thus, it is safe to take a dose for along time for the purpose of prevention.

The inventors have studied the effects on neural cell proliferation,promoting the formation of neurites and enhancing cognitive abilities byincreasing the amount of nerve growth factors in the laboratory animals,using the herb extracts mixed with Dioscorea Rhizoma and Dioscoreanipponica in a weight ratio of 3.5:1. Then, they have found thesignificant synergism compared with a single extract of DioscoreaRhizoma and Dioscorea nipponica and other ratio of them by experiencingthe laboratory animal model or cells.

Furthermore, the present invention provides a health functional food forpreventing or improving neurodegenerative disorders.

As used herein, the term “health functional food” is intended to includethose defined in the “2002 Law for health functional foods,” such ashealth foods found in the list stipulating health functional foodmaterials or ingredients verified for functionally and safety for humansaccording to KFDA Notice No. 2004-12 of the Korean Food and DrugAdministration.

More particularly, the present invention provides a health functionalfood for preventing or improving neurodegenerative disorders, comprisingthe herb extracts mixed with Dioscorea Rhizoma and Dioscorea nipponicain a weight ratio of 3.5:1 and a sitologically acceptable food additive.

The composition comprising the herb extracts mixed with DioscoreaRhizoma and Dioscorea nipponica in a weight ratio of 3.5:1 can beapplied to drugs, foods and drink for alleviating symptoms ofneurodegenerative disorders. For example, the foods to which the herbextract of the present invention can be added include beverages, gum,teas, vitamin complexes, and food aids. For use in drugs, the herbextract may be in the form of pills, powders, granules, tablets,capsules or liquids.

When applied to solid foods, the herb extract of the present inventionmay be used in an amount of food from 0.1 to 15% by weight based on thetotal weight of the food, and preferably in an amount of from 0.2 to 10%by weight. In a liquid form of health, the herb extract of the presentinvention may range in amount from 0.1 to 30 g per 100 mL of the liquidand preferably from 0.2 to 5 g.

Except for the specific ratio given to the indispensible herbingredients, no particular limitations are imparted to the liquid healthcomposition. Like typical beverages, the liquid health composition mayfurther comprise various flavoring agents, natural carbohydrates, orother additives.

Preferable examples of the natural carbohydrates include monosaccharidessuch as glucose, fructose, etc.; disaccharides such as maltose, sucrose,etc.; polysaccharides such as dextrin, cyclodextrin, etc.; and sugaralcohols such as xylitol, sorbitol, erythritol, etc. The flavoringagents useful in the present invention may be natural (thaumatin, steviaextract (e.g., rebaudioside A, glycyrrhizin), or synthetic (saccharin,aspartame). Natural carbohydrates may be used in an amount of from about1 to 20 g per 100 mL of the liquid health composition, and preferably inan amount of from about 5 to 12 g.

In addition, the composition of the present invention may containvarious nutrients, vitamins, minerals (electrolytes), synthetic and/ornatural flavoring agents, colorants, fillers (cheese, chocolate, etc.),pectic acid and its salts, alginic acid and its salts, organic acids,protective colloidal thickeners, pH modifiers, stabilizers,preservatives, glycerin, alcohols, and carbonating agents for carbonatebeverages. For use in natural fruit juice, fruit juice beverages orvegetable beverages, the composition of the present invention mayfurther contain fruit flesh. These additives may be used alone or incombination. Typical, but unimportant, amounts of the additives are onthe order of from 0 to 20 parts by weight per 100 parts by weight of thecomposition.

Advantageous Effects of Invention

The herb extracts mixed Dioscorea Rhizoma and Dioscorea nipponica in aweight ratio of 3.5:1 have the synergetic effects on increasing theamount of nerve growth factor (NGF) in vivo, increasing the neural cellproliferation, promoting the formation of neurites and enhancingcognitive abilities. Thus, the herb extracts of the present inventionmay be used for a pharmaceutical composition and health food forpreventing or improving neurodegenerative disorders.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a photograph of a hippocampus region of the brain tissueillustrating the result from the evaluation of the secretion effect ofnerve growth factor using NGF immunohistochemistry according to Example1.

MODE FOR THE INVENTION

The present invention will now be described in further detail byexamples. It would be obvious to those skilled in the art that theseexamples are intended to be more concretely illustrative and the scopeof the present invention as set forth in the appended claims is notlimited to or by the example.

Example 1

Preparation of the Mixed Herb Extracts of Dioscorea Rhizoma andDioscorea nipponica.

Dioscorea Rhizoma and Dioscorea nipponica, both in a dry condition, werepurchased from a herb medicine shop in Kyoungdong market, Korea. Afterimpurities were removed therefrom, the herbs were chopped with a cutterand mixed at a weight ratio of 3.5:1 Dioscorea Rhizoma:Dioscoreanipponica. To 2 kg of the mixture was added 10 L of a 50% ethanolsolution, followed by incubation at room temperature for 48 hours withstirring. The herb mixture was removed by filtration, and the filtratewas concentrated in a vacuum and freeze dried to afford a mixed herbextract (crude extract) (see Table 1).

TABLE 1 Yield of the mixed herb extracts Amount D. D. Amount ofExtraction Extraction Result Yield Rhizoma nipponica Solvent solventwash temperature time (g) (%) Example 1 1.55 kg 0.45 kg 50% EtOH 10 L 1L Room 2 days 212.85 10.64 Temperature

Comparative Examples 1 and 2

Preparation of Crude Herb Extracts

1. Preparation of Crude Extract of Dioscorea Rhizoma

2kg of the same Dioscorea Rhizoma used in Example 1 and stirred 2 kg ofthe same Dioscorea Rhizoma used in Example 1 and stirred 10L of 50%ethanol solution was added to for 48 hours at room temperature. Theextract of Dioscorea Rhizoma crude extract was finally obtained bylyophilization after extracting, filtering and concentrating underreduced pressure (See Table 2)

TABLE 2 Yield of the extract of Dioscorea Rhizoma Amount Amount ofExtraction Extraction Result Yield of herb Solvent solvent CleanTemperature Time (g) (%) Comparative 2 kg 50% EtOH 10 L 1 L Room 2 days253.8 12.69 Example 1 temperature

2. Preparation of Crude Extract of Dioscorea nipponica

10 L of 50% ethanol solution was added to 2 kg of the same Dioscoreanipponica used in Example 1 and stirred for 48 hour at room temperature.The extract of Dioscorea nipponnica (crude extract) was finally obtainedby lyophilization after extracting, filtering and concentrating underreduced pressure. (See table 3)

TABLE 3 Yield of the extract of Dioscorea nipponica Amount AmountExtraction Extraction Result Yield of herb Solvent of solvent WashTemperature Time (g) (%) Comparative 2 kg 50% 10 L 1 L Room 2 days 1608.00 Example 2 EtOH temperature

Comparative Examples 3 to 10

Preparation of the Mixed Herb Extract of Dioscorea Rhizoma and Dioscoreanipponica

The same herbs Dioscorea Rhizoma and Dioscorea nipponica as used inExample 1 were used. Dioscorea Rhizoma and Dioscorea nipponica werechopped with a cutter and mixed at the weight ratios listed in Table 4.To 2 kg of each of the mixtures was added 10 L of a 50% ethanolsolution, followed by incubation at room temperature for 48 hours withstirring. The herb mixtures were removed by filtration, and the filtratewas concentrated in a vacuum and freeze dried to afford mixed herbextracts (crude extracts). (See Table 4)

TABLE 4 Yield of the mixed herb extracts. Amount of herb (kg) D. D.Amount of Extraction Extraction Result Yield Rhizoma nipponica Solventsolvent Wash Temperature Time (g) (%) Comparative 1 1 50% 10 L 1 L Room2 days 179.05 8.95 Example 3 EtOH temperature Comparative 1.33 0.67 50%10 L 1 L Room 2 days 135.96 6.80 Example 4 EtOH temperature Comparative1.67 0.33 50% 10 L 1 L Room 2 days 160.07 8.00 Example 5 EtOHtemperature Comparative 1.82 0.18 50% 10 L 1 L Room 2 days 138.75 6.93Example 6 EtOH temperature Comparative 0.67 1.33 50% 10 L 1 L Room 2days 153.56 7.68 Example 7 EtOH temperature Comparative 0.45 1.55 50% 10L 1 L Room 2 days 139.15 6.96 Example 8 EtOH temperature Comparative0.33 1.67 50% 10 L 1 L Room 2 days 181.06 9.05 Example 9 EtOHtemperature Comparative 0.18 1.82 50% 10 L 1 L Room 2 days 146.43 7.32Example 10 EtOH temperature

Experimental Example 1

Effect of the Herb Extracts on Increasing the Secretion of Nerve GrowthFactor

The effects on increasing and enhancing the secretion of NGF by Example1 was identified by using C6 glioma, a cell strain of neuroglioma ofrats producing NGF. Comparative examples 1 to 10 were the control groupsof this experiment.

C6 cell was cultured in 24-well cell culture plate with medium, DMEM(adding 5% FBS, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyriuvate, 50μg streptomycin, 100 U/ml penicillin) in 37° C. and 5% of CO₂. Afteradjusting the cultured cell in the 2×10⁵ cell/well concentrations,Example 1, Comparative examples 1 to 10 were treated by 100, 250, and500 μg/ml. In the same culture condition, the cell was cultured for 2days and then the concentration of NGF in the medium was measured byusing ELISA. (See Table 5)

TABLE 5 Effects on increasing the secretion of NGF D. Rhizoma:D.nipponica Concentration compared to the total Concentration of NGFweight (μg/ml) (% of control) Example 1 3.5:1   100 122 250 149 500 156Comparative D. Rhizoma crude 100 112 Example 1 extract 250 126 500 135Comparative D. nipponica crude 100 102 Example 2 extract 250 121 500 130Comparative 1:1 100 108 Example 3 250 124 500 141 Comparative 2:1 100117 Example 4 250 131 500 136 Comparative 5:1 100 115 Example 5 250 129500 134 Comparative 10:1  100 112 Example 6 250 129 500 134 Comparative1:2 100 107 Example 7 250 116 500 129 Comparative   1:3.5 100 106Example 8 250 118 500 130 Comparative 1:5 100 107 Example 9 250 116 500126 Comparative  1:10 100 105 Example 10 250 116 500 125

As shown in Table 5, the concentration of NGF was increased according tothe concentration of Dioscorea Rhizoma and Dioscorea nipponica. Also, itwas shown that the concentration of NGF in Dioscorea Rhizoma(Comparative example 1) was more increased than in Dioscorea nipponica(Comparative example 2) so the former showed more significant effect.

Although the concentration of Dioscorea Rhizoma 25% less thancomparative example 1(D.Rhizoma 200) was injected to Example 1(D.Rhizoma:D.nipponica=155:45), 8.9 to 15.5% of NGF content wasincreased in the same concentration.

Furthermore, the concentration of NGF in Example 1 was more increasedapproximately 4.2 to 16.4% than Comparative example 5(D.Rhizoma:D.nipponica=167:33, 5:1) which Dioscorea Rhizoma was injected7.7% more and it was more increased 8.9 to 16.4% than Comparativeexample 6 (D.Rhizoma:D.nipponica=182:18, 10:1) which Dioscorea Rhizomawas injected 17.4% more). The amount of NGF in Example 1 was moreincreased approximately 4.4 to 12.9% than Comparative example 4(D.Rhizoma:D.nipponica=133:67, 2:1) which Dioscorea Rhizoma was injected14.2% less. It showed the significantly increasing content of NGF thanother Comparative examples.

Therefore it is found that a pharmaceutical composition of the presentinvention disorders comprising a mixed extract of Dioscorea Rhizoma andDioscorea nipponica in a weight ratio of 3.5:1 can increase theconcentration of NGF efficiently compared to other mixed ratio. Thus, itcould be used in treating or preventing neurodegenerative disorders.

Hereinafter, the enhancing effects on cell proliferation, formation ofneurite and cognitive abilities according to the contents of NGF in theherb extracts of Dioscorea Rhizoma and Dioscorea nipponica arecomparatively experimented. So, the availability for preventing ortreating neurodegenerative disorders will be discussed.

Experimental Example 2

Effect of the Herb Extracts on Improving Cognitive Abilities

ICR mice (male, 6 age of the weeks, 25-28 g) provided from DaehanBiolink (Chungbuk, Korea) were using after 7 days of being raised andadapted in a clean cage of college of pharmacy in Kyunghee university.They were allowed to have feeds (Daehan Biolink, Chungbuk, Korea) andwater freely. The temperature (22±2° C.), humidity (53±3%) and cycle oflight and dark (12 hours) were controlled automatically.

10 mice were assigned to each group. Saline was orally administered tomice in the control group in 5 mL per kg of weight. 10, 100 mg/kg ofExample 1, Comparative examples 1 to 10 respectively melted in salinesolution were orally administered to mice in the treatment group. Oraladministration was done once a day and 1 hour after the last oraladministration, passive avoidance test were conducted as follows.

Passive avoidance test was conducted in the A modified shuttle with twocommunicating (7×7 cm sliding door built into the separating wall)compartments of equal size and a stainless steel bar floor was used. Theright-hand compartment (shock compartment) was painted black to obtain adark chamber. The left-hand compartment was illuminated by a bulb (24 V;5 W) installed on the top Plexiglas cover. First day, a light turns onin a bright room and turns off in a dark room. And the inventors openedguillotine door after letting mice stay in a bright room for 10 secondsthen measured the time until mice entered a dark room. When mice movedinto a dark room, guillotine door closed and 0.3 mA of electricalstimulation was given to them for 3 seconds. After 24 hours, passiveavoidance test was demonstrated with the same method and latency time ofstaying in a bright room was measured. The results are shown in Table 6.

TABLE 6 Effect on improving cognitive abilities in passive avoidancetest D. Rhizoma:D. nipponica compared to the total Amount Latency Timeweight (mg/kg) (sec) Control Group 166.18 ± 13.83 Example 1 3.5:1   10220.17 ± 25.63 100 275.87 ± 25.40 Comparative D. Rhizoma crude extract10 202.84 ± 19.65 Example 1 100 225.28 ± 20.21 Comparative D. nipponicacrude extract 10 199.62 ± 17.01 Example 2 100 218.24 ± 18.47 Comparative1:1 10 194.86 ± 9.43  Example 3 100 204.19 ± 10.72 Comparative 2:1 10202.69 ± 11.24 Example 4 100 231.42 ± 16.55 Comparative 5:1 10 211.43 ±18.21 Example 5 100 235.71 ± 21.60 Comparative 10:1  10 205.33 ± 15.08Example 6 100 219.93 ± 16.14 Comparative 1:2 10 186.72 ± 1061  Example 7100 204.68 ± 14.32 Comparative   1:3.5 10 196.93 ± 16.58 Example 8 100209.61 ± 14.41 Comparative 1:5 10 205.89 ± 12.60 Example 9 100 215.99 ±18.96 Comparative  1:10 10 193.53 ± 16.08 Example 10 100 211.74 ± 20.57

As shown in Table 6, the retention latency in the Dioscorea Rhizomaadministrating group was increased than that of Dioscorea nipponicaadministrating group, Example 1 (D.Rhizoma:D.nipponica=155:45) increasedretention latency 8.9 to 22.2% than Comparative example 1 (D.Rhizoma200), although Example 1 was administered 25% less Dioscorea Rhizomathan Comparative example 1 (D.Rhizoma 200).

Also, the retention latency according to Example 1 was increased 4.2 to19% than Comparative example 5 (D.Rhizoma:D.nipponica=167:33, 5:1) whichDioscorea Rhizoma was injected 7.7% more and it was more increased 7.3to 25.6% than Comparative example 6 (D.Rhizoma:D.nipponica=182:18, 10:1)which Dioscorea Rhizoma was injected 17.4% more). The retention latencyaccording to Example 1 was increased 8.9 to 19% than Comparative example4 (D.Rhizoma:D.nipponica=133:67, 2:1) which Dioscorea Rhizoma wasinjected 14.2% less. It showed the memory was increased significantlythan other Comparative examples.

Therefore it is found that a pharmaceutical composition of the presentinvention disorders comprising a mixed extract of Dioscorea Rhizoma andDioscorea nipponica in a weight ratio of 3.5:1 has the effects onimproving the cognitive abilities compared to other mixed ratio.

Experimental Example 3

Effect the Herb Extracts on Enhancing Generation of Neurons

Hippocampi were separated from mice of each group after completing thetest of Experimental example 2. 5-bromo-2-deoxyuridine (BrdU, santacruz, rat origin 1:500) as a first antibody was reacted for one nightafter dehydrating separated hippocampus with hydrogen peroxide. Andthen, biotinylated anti-rat (vector, goat origin) was used as a secondantibody, and color was developed by using Diaminobenzidine after ABCreaction (ABC kit, vector). The effect on enhancing neuronal cellgeneration was identified by counting BrdU-positive cells in the area ofdentate gyrus in hippocampus. The results are shown in Table 7.

TABLE 7 Effect on enhancing cell generation D. Rhizoma:D. nipponicacompared to the total Amount BrdU-positive cells weight (mg/kg) (% ofcontrol) Example 1 3.5:1   10 153.79 ± 15.24 100 158.59 ± 9.45Comparative D. Rhizoma crude extract 10 123.81 ± 10.14 Example 1 100129.42 ± 11.36 Comparative D. nipponica crude extract 10 118.60 ± 8.37Example 2 100 121.59 ± 8.13 Comparative 1:1 10 124.25 ± 10.93 Example 3100 125.86 ± 9.06 Comparative 2:1 10 128.44 ± 11.65 Example 4 100 136.70± 13.28 Comparative 5:1 10 129.54 ± 12.91 Example 5 100 134.08 ± 14.61Comparative 10:1  10 121.08 ± 9.54 Example 6 100 128.57 ± 11.77Comparative 1:2 10 120.68 ± 9.27 Example 7 100 124.16 ± 11.36Comparative   1:3.5 10 126.39 ± 11.17 Example 8 100 130.28 ± 13.49Comparative 1:5 10 124.70 ± 10.25 Example 9 100 128.06 ± 11.59Comparative  1:10 10 121.84 ± 13.18 Example 10 100 124.12 ± 12.85

As shown in Table 7, Dioscorea Rhizoma (Example 1) showed increased thenumber of BrdU-positive cells than Dioscorea nipponica (Comparativeexample 2) when counting BrdU-positive cells in the area of dentategyrus in hippocampus. Although the amount of Dioscorea Rhizoma wasinjected to Example 1 25% less than Comparative example 1, the number ofBrdU-positive cells were increased approximately 25% more.

Furthermore, the number of BrdU-positive cells were increasedapproximately 25% more in Example 1 than Comparative example 5 whichDioscorea Rhizoma was injected 7.7% more than Example 1 and Comparativeexample 6 which Dioscorea Rhizoma was injected 17.4% more thanExample 1. The number of BrdU-positive cells were increasedapproximately 20% more in Example 1 than Comparative example 4 whichDioscorea Rhizoma was injected 14.2% less than Example 1. It showed thesignificantly increasing the number of BrdU-positive cells than otherComparative examples.

Therefore it is found that a pharmaceutical composition of the presentinvention disorders comprising a mixed extract of Dioscorea Rhizoma andDioscorea nipponica in a weight ratio of 3.5:1 has the effects onenhancing the neuronal cell generation in the area of dentate gyruscompared to other mixed ratio.

Experimental Example 4

Effect of the Herb Extracts on Neuronal Cell Differentiation andEnhancing Neurite Growth

Hippocampi were separated from mice of each group after completing thetest of Experimental example 2. Doublecortin (DCX, santa cruz, goatorigin 1:500) as a first antibody was reacted for one night afterdehydrating separated hippocampus with hydrogen peroxide. And then,biotinylated anti-goat (vector, horse origin) was used as a secondantibody, and color was formulated by using Diaminobenzidine after ABCreaction (ABC kit, vector). The effect on cell differentiation andenhancing neurite growth was identified by counting DCX-positive cellsin the area of dentate gyrus in hippocampus. The results are shown inTable 8.

TABLE 8 Effect on cell differentiation and enhancing neurite growth D.Rhizoma:D. nipponica compared to the total Amount DCX-positive cellLength of neurite weight (mg/kg) (% of control) (% of control) Example 13.5:1   10 156.24 ± 9.40 125.38 ± 2.82 100 183.04 ± 12.67 142.59 ± 1.66Comparative D. Rhizoma crude 10 126.84 ± 7.05 115.42 ± 3.60 Example 1extract 100 132.81 ± 9.69 121.34 ± 2.56 Comparative D. nipponica crude10 118.60 ± 8.37 120.48 ± 3.74 Example 2 extract 100 121.59 ± 8.13120.57 ± 2.36 Comparative 1:1 10 127.19 ± 7.23 120.48 ± 3.74 Example 3100 132.16 ± 9.61 129.63 ± 2.16 Comparative 2:1 10 130.80 ± 8.67 124.08± 2.48 Example 4 100 141.64 ± 7.85 131.75 ± 1.81 Comparative 5:1 10132.54 ± 10.34 121.64 ± 1.34 Example 5 100 144.10 ± 9.32 130.12 ± 2.73Comparative 10:1  10 127.06 ± 11.46 123.39 ± 1.89 Example 6 100 138.42 ±9.29 129.61 ± 2.48 Comparative 1:2 10 125.47 ± 14.38 119.42 ± 1.99Example 7 100 131.37 ± 11.94 126.74 ± 2.65 Comparative   1:3.5 10 126.88± 10.43 120.74 ± 1.84 Example 8 100 133.05 ± 9.59 122.38 ± 1.69Comparative 1:5 10 130.15 ± 9.29 125.42 ± 2.88 Example 9 100 135.53 ±10.74 131.41 ± 2.15 Comparative  1:10 10 124.06 ± 9.86 121.72 ± 1.61Example 10 100 126.91 ± 10.34 124.63 ± 2.83

As shown in Table 8, in the area of dentate gyrus in hippocampus,Dioscorea Rhizoma (Example 1) showed increased the number ofDCX-positive cells and the length of DCX-positive neurites thanDioscorea nipponica (Comparative example 2). Although the amount ofDioscorea Rhizoma was injected to Example 1 25% less than Comparativeexample 1, the number of DCX-positive cells and the length ofDCX-positive neurites were increased approximately 25 to 40% and 1.6 to17.3% more, respectively.

Furthermore, the number of DCX-positive cells and the length ofDCX-positive neurites were increased approximately 18 to 27% and 3.3 to9.2% more, respectively in Example 1 than Comparative example 5(Dioscorea Rhizoma was injected 7.7% more than Example 1). The number ofDCX-positive cells and the length of DCX-positive neurites wereincreased approximately 22.8 to 32.6% and 3.3 to 10% more, respectivelyin Example 1 than Comparative example 6 (Dioscorea Rhizoma was injected17.4% more than Example 1). The number of DCX-positive cells and thelength of DCX-positive neurites were increased approximately 20 to 30%and 1 to 8% more, respectively in Example 1 than Comparative example 4(Dioscorea Rhizoma was injected 14.2% less than Example 1). It showedthe significantly increasing the number of DCX-positive cells and thelength of DCX-positive neurites than other Comparative examples.

Therefore it is found that a pharmaceutical composition of the presentinvention disorders comprising a mixed extract of Dioscorea Rhizoma andDioscorea nipponica in a weight ratio of 3.5:1 has the effects onneuronal cell differentiation and enhancing neurite growth in the areaof dentate gyrus compared to other mixed ratio.

Experimental Example 5

Effect of the Herb Extracts on Enhancing the Secretion of NGF in Vivo

Hippocampi were separated from mice of the control group and thetreatment group which was injected the mixed extract(D.Rhizoma:D.nipponica=3.5:1 extract) after completing the test ofExperimental example 2. Anti-Nerve growth factor (NGF, abcam, rabbitorigin 1:500) as a first antibody was reacted for one night and thenbiotinylated anti-rat (vector, goat origin) was used as a secondantibody. After that, fluorescene-color was formulated by usingstreptavidin after ABC reaction (ABC kit, vector). The effect onenhancing the secretion of NGF was identified by counting NGF-positivecells in the area of hilus in hippocampus. The results are shown in FIG.1.

As shown in FIG. 1, the herb extracts of Example 1 has the significanteffect on enhancing the secretion of NGF in hippocampus.

Therefore it is found that a pharmaceutical composition of the presentinvention comprising a mixed extract of Dioscorea Rhizoma and Dioscoreanipponica in a weight ratio of 3.5:1 has the synergism compared to othermixed ratio. Thus, a pharmaceutical composition of the present inventionmay be used for preventing or treating neurodegenerative disordersincluding Alzheimer's disease, Creutzfeldt-Jakob disease, Huntington'sdisease, multiple sclerosis, Guillain-Barre syndrome, Parkinson'sdisease, Lou Gehrig's disease, paralytic dementia caused by gradualnerve cell death, diseases caused by progressive incotinentia,abnormalities in posture and exercise, progressive ataxia, muscularatrophy and weakness, and sensation and movement disorders.

Now, the composition comprising the herb extracts disclosed in Example 1will described in further detail by Formulation Examples. There havebeen no intentions to limit the claims but explain specifically.

Formulation Example 1. Preparation of Injection

Extract of Example 1 100 mg  Sodium metabisulfite 3.0 mg Methylparaben0.8 mg Propylparaben 0.1 mg Sterile water for injection q.s.

To a mixture of the ingredients was added sterile water to form a totalvolume of 2 mL, and the solution was loaded to a 2 mL ampule andsterilized to give an injection.

Formulation Example 2. Preparation of Tablet

Extract of Example 1 200 mg Lactose 100 mg Starch 100 mg Mg stearateq.s.

The ingredients were mixed and compressed into a tablet using atableting method.

Formulation Example 3. Preparation of Capsule

Extract of Example 1 100 mg  Lactose 50 mg Starch 50 mg Talc  2 mg MgStearate q.s.

The ingredients were mixed and loaded to a gelatin capsule according toa typical method to afford a capsule.

Formulation Example 4. Preparation of Liquid.

Extract of Example 1 1000 mg sugar  20 g Isomeroase  20 g Lemon Flavorq.s.

Purified water added to form a total volume of 100 mL

The above ingredients were mixed, loaded into a 100 mL brown vial andsterilized to afford a liquid formulation.

1. A pharmaceutical composition for preventing or treatingneurodegenerative disorders, comprising: mixed herb extracts ofDioscorea Rhizoma and Dioscorea nipponica in a weight ratio of 3.5:1(w/w) as an active ingredient; and a pharmaceutically acceptablecarrier.
 2. The pharmaceutical composition for preventing or treatingneurodegenerative disorders of claim 1, wherein the neurodegenerativedisorder is selected from the group consisting of Alzheimer's disease,Creutzfeldt-Jakob disease, Huntington's disease, multiple sclerosis,Guillain-Barre syndrome, Parkinson's disease, Lou Gehrig's disease,paralytic dementia caused by gradual nerve cell death and diseasescaused by progressive incontinentia.
 3. A health functional foodcomposition for preventing or improving neurodegenerative disorders,comprising mixed herb extracts of Dioscorea Rhizoma and Dioscoreanipponica in a weight ratio of 3.5:1 (w/w).
 4. The health functionalfood composition of claim 3, wherein the herb extracts are the crudeextracts extracted by using 50% of ethanol.
 5. The health functionalfood composition of claim 3, wherein the herb extracts are prepared intoa form selected from the group consisting of a powder, a granule, atablet, a capsule, a syrup and a beverage.